Water soluble anionic bacteriochlorophyll derivatives and their uses

ABSTRACT

The invention provides anionic water-soluble tetracyclic and pentacyclic bacteriochlorophyll derivatives (Bchls) containing at least one, preferably two or three, negatively charged groups and/or acidic groups that are converted to negatively charged groups at the physiological pH, preferably Bchls having a group COO&lt;−&gt;, COS&lt;−&gt;, SO3&lt;−&gt;, PO3&lt;2−&gt;, COOH, COSH, SO3H, and/or PO3H2 bound through an ester or amide bond to one or more of the positions 17&lt;3&gt;, 13&lt;3&gt;, and 3&lt;2&gt; of the tetracyclic or pentacyclic Bchl molecule, for photodynamic therapy and diagnosis.

FIELD OF THE INVENTION

The present invention relates to novel water-soluble anionic derivativesof bacteriochlorophyll, to their preparation and their use in methods ofin-vivo photodynamic therapy and diagnosis of tumors and differentvascular diseases such as age-related macular degeneration, as well asin methods of in-vivo and ex-vivo killing of viruses and microorganisms.

DEFINITIONS AND ABBREVIATIONS

AMD: age-related macular degeneration;Bchl: bacteriochlorophyll a—pentacyclic 7,8,17,18-tetrahydroporphyrinwith a 5^(th) isocyclic ring, a central Mg atom, a phytyl orgeranylgeranyl group at position 17³, a COOCH₃ group at position 13², anH atom at position 13², methyl groups at positions 2, 7, 12, 18, anacetyl group at position 3, and an ethyl group at position 8;Bphe: bacteriopheophytin a (Bchl in Which central Mg is replaced by twoH atoms);Bpheid: bacteriopheophorbide a (the C-17²-free carboxylic acid derivedfrom BPhe);

Pd-Bpheid: Pd-bacteriopheophorbide a;

PDT: photodynamic therapy;Rhodobacteriochlorin: tetracyclic 7,8,17,18-tetrahydroporphyrin having a—CH₂CH₂COOH group at position 17, a —COOH at position 13, methyl groupsat positions 2; 7, 12, 8, and ethyl groups at positions 3 and 8.

IUPAC numbering of the bacteriochlorophyll derivatives is usedthroughout the specification. Using this nomenclature, the naturalbacteriochlorophylls carry two carboxylic acid esters at positions 13²and 17², however they are esterified at positions 13³ and 17³.

BACKGROUND OF THE INVENTION

Photo dynamic therapy (PDT) is a non-surgical treatment of tumors inwhich non-toxic drugs and non-hazardous photosensitizing irradiation arecombined to generate cytotoxic reactive oxygen species in situ. Thistechnique is more selective than the commonly used tumor chemotherapyand radiotherapy. To date, porphyrins have been employed as the primaryphotosensitizing agents in clinics. However, current sensitizers sufferfrom several deficiencies that limit their application, includingmainly: (1) relatively weak absorption in the visible spectral rangewhich limits the treatment to shallow tumors; (2) accumulation and longretention of the sensitizer in the patient skin, leading to prolonged(days to months) skin phototoxicity; and (3) small or even nodifferentiation between the PDT effect on illuminated tumor andnon-tumor tissues. The drawbacks of current drugs inspired an extensivesearch for long wavelength absorbing second-generation sensitizers thatexhibit better differentiation between their retention in tumor cellsand skin or other normal tissues.

In order to optimize the performance of the porphyrin drugs intherapeutics and diagnostics, several porphyrin derivatives have beenproposed in which, for example, there is a central metal atom (otherthan Mg) complexed to the four pyrrole rings, and/or the peripheralsubstituents of the pyrrole rings are modified and/or the macrocycle isdihydrogenated to chlorophyll derivatives (chlorins) ortetrahydrogenated to bacteriochlorophyll derivatives (bacteriochlorins).

Due to their intense absorption in favorable spectral regions (650-850nm) and their ready degradation after treatment, chlorophyll andbacteriochlorophyll derivatives have been identified as excellentsensitizers for PDT of tumors and to have superior properties incomparison to porphyrins, but they are less readily available and moredifficult to handle.

Bacteriochlorophylls are of potential advantage compared to thechlorophylls because they show intense near-infrared bands, i.e. atconsiderably longer wavelengths than chlorophyll derivatives.

The spectra, photophysics, and photochemistry of nativebacteriochlorophylls (Bchls) have made them optimal light-harvestingmolecules with clear advantages over other sensitizers presently used inPDT. In particular, these molecules have a very high extinctioncoefficient at long wavelengths (λ_(max)=760-780 nm, ε=(4-10)×10⁴ cm⁻¹),where light penetrates deeply into tissues. They also generate reactiveoxygen species (ROS) at a high quantum yield (depending on the centralmetal).

Under normal delivery conditions, i.e. in the presence of oxygen at roomtemperature and under normal light conditions, the BChl moieties arelabile and have somewhat lower quantum yields for triplet stateformation, when compared with, e.g., hematoporphyrin derivative (HPD).However, their possible initiation of biological redox reactions,favorable spectral characteristics and their ready degradation in vivoresult in the potential superiority of bacteriochlorophylls over othercompounds, e.g. porphyrins and chlorophylls, for PDT therapy anddiagnostics and for killing of cells, viruses and bacteria in samplesand in living tissue. Chemical modification of bacteriochlorophylls isexpected to further improve their properties, but this has been verylimited due to lack of suitable methods for the preparation of suchmodified bacteriochlorophylls.

The biological uptake and PDT efficacy of metal-free derivatives of Bchlhave been studied with the objective to manipulate the affinity of thesensitizers to the tumor cellular compartment. Cardinal to this approachis the use of highly lipophilic drugs that may increase the accumulationof the drug in the tumor cells, but also renders its delivery difficult.In addition, the reported biodistribution shows significant phototoxicdrug levels in non-tumor tissues over prolonged periods (at least days)after administering the drug.

In applicant's previous Israel Patent No. 102645 and corresponding EP0584552, U.S. Pat. No. 5,726,169, U.S. Pat. No. 5,726,169, U.S. Pat. No.5,955,585 and U.S. Pat. No. 6,147,195, a different approach was taken bythe inventors. Highly efficient anti-vascular sensitizers that do notextravasate from the circulation after administration and have shortlifetime in the blood were studied. It was expected that the inherentdifference between vessels of normal and abnormal tissues such as tumorsor other tissues that rely on neovessels, would enable relativelyselective destruction of the abnormal tissue. Hence, it was aimed tosynthesize Bchl derivatives that are more polar and, hence, have betterchance to stay in the vascular compartment, where they convey theprimary photodynamic effect. To this end, the geranylgeranyl residue atthe C-17 position of Bchl a (Compound 1, depicted in Scheme 1 herein)has been replaced by various residues such as amino acids, peptides, orproteins, which enhance the sensitizer hydrophilicity. One particularderivative, Bchl-Ser (Scheme 1, Compound 1, wherein R is seryl), wasfound to be water-soluble and highly phototoxic in cell cultures.Following intraperitoneal injection, the Bchl-Ser cleared from the mouseblood and tissues bi-exponentially in a relatively short time (t_(1/2)˜2 and 16 h, respectively). Clearance from the circulation was evenfaster following intravenous injection. Under the selected treatmentprotocol (light application within minutes after drug injection),phototoxicity was predominantly conferred to the tumor vasculature(Rosenbach-Belkin et al., 1996; Zilberstein et al., 2001 and 1997).However, unfortunately, like native Bchl, the Bchl-Ser derivativeundergoes rapid photo-oxidation, forming the corresponding2-desvinyl-2-acetyl-chlorophyllide ester and other products.

To increase the stability of the Bchl derivatives, the central Mg atomwas replaced by Pd in the later applicant's PCT Publication WO 00/33833and U.S. Pat. No. 6,569,846. This heavy atom was previously shown tomarkedly increase the oxidation potential of the Bchl macrocycle and, atthe same time, to greatly enhance the intersystem-crossing (ISC) rate ofthe molecule to its triplet state. The metal replacement was performedby direct incorporation of Pd²⁺ ion into a Bpheid molecule, as describedin WO 00/33833. Based on the pigment biodistribution andpharmacokinetics, it was assumed that the derivative Pd-Bpheid remainedin the circulation for a very short time with practically noextravasation to other tissues, and is therefore a good candidate forvascular-targeting PDT that avoids skin phototoxicity. The treatmenteffect on the blood vessels was demonstrated by intravital microscopy oftreated blood vessels and staining with Evans-Blue. Using a treatmentprotocol with a minimal drug-to-light interval, Pd-Bpheid (alsodesignated Tookad) was found to be effective in the eradication ofdifferent tumors in mice, rats and other animal models and is presentlyentering Phase I/II clinical trials in patients with prostate cancerthat failed radiation therapy (Chen et al., 2002; Schreiber et al.,2002; Koudinova et al., 2003).

Because of its low solubility in aqueous solutions, the clinical use ofPd-Bpheid requires the use of solubilizing agents such as Cremophor thatmay cause side effects at high doses. It would be highly desirable torender the Pd-Bpheid water-soluble while retaining its physico-chemicalproperties. Alternatively, it would be desirable to prepare Bchlderivatives that are cytophototoxic and, at the same time, morewater-soluble than Pd-Bpheid itself. Such water solubility is expectedto further enhance the drug retention in the circulation and, thereby,the aforementioned selectivity. In addition, having no need to usecarriers such as detergents or lyposomes, may prevent side effects.

SUMMARY OF THE INVENTION

The present invention relates, to a bacteriochlorophyll derivativecontaining at least one, preferably two or three, negatively chargedgroups and/or acidic groups that are converted to negatively chargedgroups at the physiological pH, excluding pentacyclicbacteriochlorophyll derivatives having a free CH₂CH₂COOH or a CH₂CH₂COO⁻group at position 17, and tetracyclic bacteriochlorophyll derivativesdevoid of a central metal atom and having a —CH₂CH₂COOH group atposition 17, a —CH₂COOH or COOH group at position 15, a —COOH group atposition 13, methyl groups at the positions 2, 7, 12, 18, and ethylgroups at the positions 3 and 8.

The negatively charged groups according to the invention include, butare not limite to, carboxylate (COO⁻), thiocarboxylate (COS⁻), sulfonate(SO₃ ⁻), and phosphonate (PO₃ ²⁻), and the acidic groups from which saidcharged groups originate at the physiological pH are the carboxylic(COOH), thiocarboxylic (COSH), sulfonic (SO₃H) and phosphoric (PO₃H₂)acid groups, respectively.

In one embodiment, the bacteriochlorophyll derivative has the formula Ior II:

wherein

M represents 2H or a metal atom selected from the group consisting ofdivalent Pd, Pt, Co, Sn, Ni, Cu, Zn and Mn, and trivalent Fe, Mn and Cr;

R₁, R₂, and R₄ each independently is Y—R₅;

-   -   Y is O, S or NR₅R₆;

R₃ is selected from the group consisting of CH═CH₂, —C(═O)—CH₃, —CH═NR₇,—C(CH₃)═NR₇, —CH₂—OR₇, —CH₂—SR₇, —CH₂—NR₇R′₇, —CH(CH₃)—OR₇,—CH(CH₃)—SR₇, —CH(CH₃)—NR₇R′₇, —CH(CH₃)Hal, —CH₂—Hal, —CH₂—R₇,—CH═CR₇R′₇, —C(CH₃)═CR₇R′₇, —CH═CR₇Hal, —C(CH₃)=CR₇Hal, and —C≡CR₇;

R₅, R₆, R₇ and R′₇ each independently is H or selected from the groupconsisting of:

(a) C₁-C₂₅ hydrocarbyl optionally containing one or more heteroatoms,carbocyclic or heterocyclic moieties, and/or optionally substituted byone or more functional groups selected from the group consisting ofhalogen, oxo, OH, SH, CHO, NH₂, CONH₂, a negatively charged group, andan acidic group that is converted to a negatively charged group at thephysiological pH;

(b) a residue of an amino acid, a peptide or of a protein; and

(c) when Y is O or S, R₅ may further be R₈ ⁺;

M is 0 or 1; and

R₈ ⁺ is H⁺ or a cation;

provided that:

(i) at least one, preferably two, of R₅, R₆, R₇ and R′₇ is a hydrocarbonchain as defined in (a) above substituted by a negatively charged groupor by an acidic group that is converted to a negatively charged group atthe physiological pH; or

(ii) at least one, preferably two, of R₁, R₂, and R₄ is OH, SH, O⁻R₈ ⁺or SR₈ ⁺;

(iii) at least one of R₁, R₂, and R₄ is OH, SH, O⁻R₈ ⁺ or S⁻R₈ ⁺ and atleast one of R₅, R₆, R₇ and R′₇ is a hydrocarbon chain substituted by anegatively charged group or by an acidic group that is converted to anegatively charged group at the physiological pH; or

(iv) at least one of R₁, R₂, and R₄ is OH, SH, O⁻ R₈ ⁺ or S R₈ ⁺ and atleast one of R₅, R₆, R₇ and R′₇ is a residue of an amino acid, a peptideor of a protein; or

(v) at least one of R₅, R₆, R₇ and R′₇ is a hydrocarbon chainsubstituted by a negatively charged group or by an acidic group that isconverted to a negatively charged group at the physiological pH and atleast one of R₅, R₆, R₇ and R′₇ is a residue of an amino acid, a peptideor of a protein;

but excluding the compounds of formula I wherein M is as defined, R₃ is—C(═O)CH₃, R₁ is OH or OR₈ ⁺ and R₂ is —OCH₃, and the compound offormula II wherein M is 2H, R₃ is —C(═O)CH₃, R₁, R₂ and R₄ are OH, and mis 0 or 1.

The invention further relates to pharmaceutical compositions comprisinga bacteriochlorophyll derivative as defined above for photodynamictherapy (PDT), particularly for vascular-targeting PDT, for example forPDT of tumors or of age-related macular degeneration (AMD), or forkilling cells or infectious agents comprising bacteria and viruses invivo or in vitro, as well as for diagnostic purposes.

The invention provides a method for photodynamic therapy using aphotosensitizer, wherein the improvement consists in that saidphotosensitizer is a bacteriochlorophyll derivative of the invention.According to this aspect, the invention relates to a method fortreatment by PDT which comprises administering to an individual in needan effective amount of a bacteriochlorophyll derivative of theinvention, followed by local irradiation.

The invention further provides a method for diagnosis of tumors using aphotosensitize; wherein the improvement consists in that saidphotosensitizer is a bacteriochlorophyll derivative of the invention.According to this aspect, the invention relates to a method fordiagnosis of tumors which comprises administering to an individualsuspected of having a tumor an effective amount of a bacteriochlorophyllderivative of the invention, followed by local irradiation and measuringthe fluorescence of the suspected area, wherein a higher fluorescenceindicates tumor sites.

The invention still further provides a method for killing cells orinfectious agents comprising bacteria and viruses, using aphotosensitize; the improvement wherein said photosensitizer is abacteriochlorophyll derivative of the invention. According to thisaspect, the invention relates to a method for sterilization ofbiological products, e.g. blood, which comprises adding to saidbiological product, e.g. blood, an effective amount of abacteriochlorophyll derivative of the invention, followed byirradiation.

BRIEF DESCRIPTION OF THE FIGURES

The different compounds of the invention are represented in thefollowing description of the drawings by a bold and underlined numeral.Their full identification is found in the List of Compounds at thebeginning of the Chemical Section hereinafter.

FIGS. 1A-1B are graphs showing the phototoxicity of the sulfonatedcompound 8 on H5V mouse endothelial cells (FIG. 1A) and M2R mousemelanoma cells (FIG. 1B). Cells were incubated with increasingconcentrations of 8 for 4 hours, washed and illuminated (open shapes) orkept in the dark (dark control, closed shapes). Points are average oftriplicates ±STD.

FIGS. 2A-2B are graphs showing the phototoxicity of the sulfonatedcompound 4 on H5V mouse endothelial cells (FIG. 2A) and M2R mousemelanoma cells (FIG. 2B). Cells were incubated with increasingconcentrations of compound 4 for 4 hours, washed and illuminated (openshapes) or kept in the dark (dark control, closed shapes). Points areaverage of triplicates ±STD.

FIG. 3 is a graph showing the phototoxicity of the sulfonated compound 5on M2R mouse melanoma cells. Cells were incubated with increasingconcentrations of compound 5 for 4 hours, washed and illuminated(circles) or kept in the dark (dark control, diamonds). Points areaverage of triplicates.

FIG. 4 is a graph showing the phototoxicity of the sulfonated compound11 on M2R mouse melanoma cells. Cells were incubated with increasingconcentrations of compound 11 for 4 hours, washed and illuminated(circles) or kept in the dark (dark control, diamonds). Points areaverage of triplicates.

FIG. 5 is a graph showing pharmacokinetics of compound 4 in CD1 nudemice blood. Following compound 4 injection (6 mg/kg), blood samples werecollected from the same mouse at the indicated times and Pd wasdetermined. Each time point represents average of three mice ±STD.

FIG. 6 shows biodistribution of compound 4 in CD1 nude mice. Mice weresacrificed at different times following compound 4 injection (6 mg/kg),and Pd content was determined for the indicated organs. Each time pointrepresents' average of three mice ±STD.

FIG. 7 shows PDT of melanoma xenografts with compound 4. Mice bearingM2R melanoma xenografts were intravenously injected with compound 4 (6mg kg⁻¹) and illuminated for 5 min with light intensity of 30 J/cm²(n=14, filled squares), 39 J/cm² (n=8, filled diamonds) or 45 J/cm²(n=10, filled triangles). Mice that were injected with 9 mg kg⁻¹ ofcompound 4 were illuminated for 5 min with 30 J/cm² (n=10, filledcircles). Control groups: untreated (n=4, open squares), dark controlreceived 6 mg kg⁻¹ (n=4, open circles) or 9 mg kg⁻¹ (n=5, opentriangles) of compound 4, and light control (n=6, open diamonds,453/cm²).

FIGS. 8a-8h are photographs showing the selective effect of PDT in micebearing rat C6 glioma xenografts and treated with compound 4. (a-d) PDTtreated animal; (e-h) untreated animal. (a) before treatment; (b) 3hours after PDT and Evans-Blue (EB) injection; (c) skin flap of thetreated area, 24 hours after PDT; (d) axial slice of the treated tumor24 hours after PDT; (e) before EB injection; (f) 3 hours after EBinjection; (g) skin flap 24 hours after EB injection; (h) axial slice ofthe untreated tumor, 24 hours after EB injection. T-tumor; S-skin;M-muscle; E-edema.

FIGS. 9A-9D show semi-thin sections of the lesion center and TEM 2 hoursafter occlusive PDT in a rabbit eye model with compound 4 (fluence 50J/cm2, dose of 5 mg/Kg, and a DLI of 1 minute). Stasis and dilatation ofchoroidal vessels with relatively well preserved RPE cells and retinaare observed (9A and 9B). TEM shows hemolysis of the red blood cellswithin the choriocapillary lumen (white arrows of 9D) and disruptedmonocytes (white arrowhead). Bruch's membrane (Bm) is intact harboringwell identified retinal pigment epithelium cells (RPE). Some of thechoriocapillary endothelial cells are markedly altered demonstratingcondensed chromatin (white star on 9C). Abbreviations: ONL: outernuclear layer, ROS: rod outer segments, CC: Choriocappilaries, e:choriocapillary endothelial cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in a broad aspect, bacteriochlorophyllderivatives containing at least one, preferably two or three, negativelycharged groups and/or acidic groups that are converted to negativelycharged groups at the physiological pH, excluding pentacyclicbacteriochlorophyll derivatives having a free —CH₂CH₂COOH or —CH₂CH₂COO⁻group at position 17, and tetracyclic bacteriochlorophyll derivativesdevoid of a central metal atom and having a —CH₂CH₂COOH group atposition 17, a —CH₂COOH or COOH group at position 15, a —COOH group atposition 13, a methyl group at each of the positions 2, 7, 12, and 18,and an ethyl group at each of the positions 3 and 8.

The bacteriochlorophyll derivatives may be derived from a natural orsynthetic derivative of bacteriochlorophyll, including compounds inwhich the central Mg atom has been deleted or replaced by other metalatoms such as divalent Pd, Pt, Co, Sn, Ni, Cu, Zn and Mn, and trivalentFe, Mn and Cr. In preferred embodiments, the metal atom is absent or itis Pd, Cu, Zn or Mn. In the most preferred embodiment, the central metalatom is Pd.

In one preferred embodiment, the present invention provides abacteriochlorophyll derivative of the formula I or II as definedhereinabove.

According to the invention, “hydrocarbyl” as defined for R₅, R₆, R₇ andR′₇ means any straight or branched, saturated or unsaturated, acyclic orcyclic, including aromatic, hydrocarbyl radicals, of 1-25 carbon atoms,preferably of 1 to 20, more preferably 1 to 6, most preferably 2-3carbon atoms. The hydrocarbyl may be an alkyl radical, preferably of 1-4carbon atoms, e.g. methyl, ethyl, propyl, butyl, or alkenyl, alkynyl,cycloalkyl, aryl such as phenyl or an aralkyl group such as benzyl, orat the position 17 it is a radical derived from natural Chl and Milcompounds, e.g. geranylgeranyl (2,6-dimethyl-2,6-octadienyl) or phytyl(2,6,10,14-tetramethyl-hexadec-14-en-16-yl).

The hydrocarbon chain of R₅, R₆, R₇ and/or R′₇ may optionally containone or more heteroatoms such as O, S and/or NH, and/or one or morecarbocyclic ring, e.g. phenyl, or heterocyclic ring, e.g pyridyl,moieties. In one embodiment, the hydrocarbyl chain contains one or moreO atoms and has a OH end group as represented by anoligooxyethyleneglycol residue of 4 to 10 carbon atoms, preferablypentaoxyethyleneglycol.

R₅, R₆, R₇ and/or R′₇ may also be hydrocarbyl substituted by one or morefunctional groups, such as Cl, CHO, OH, SH, NH₂, CONH₂, COOH, COSH,SO₃H, PO₃H₂ or by a negatively charged group such as COO⁻, COS⁻, SO₃ ⁻,or PO₃ ²⁻. In one preferred embodiment, the functional group COOH, COSH,SO₃H, PO₃H₂, COO⁻, COS⁻, SO₃ ⁻, or PO₃ ²⁻ is an end functional group. Inmost preferred embodiments, the hydrocarbyl has 2 or 3 carbon atoms andan end group selected from COO⁻, PO₃ ²⁻, or, most preferably, SO₃ ⁻.

In still a further embodiment, R₅, R₆, R₇ or R′₇ may be substituted bymore than one OH and optionally NH₂ groups and may be the residue of amonoaccharide, e.g., glucosamine.

In another embodiment, R₅, R₆, R₇ or R′₇ may be the residue of an aminoacid, a peptide or a protein. In one preferred embodiment, R₅ at any ofthe positions, but preferably at position 17³, is the residue of anamino acid, a peptide or a protein. The amino acid, peptide or proteinmay be the source of the negatively charged group if they contain a freeterminal carboxyl group and/or a residue of an amino acid containing anon-terminal free carboxylic group, e.g. aspartic or glutamic acid.

In one embodiment, R₅, R₆, R₇ or R′₇ is the residue of an amino acid orpeptide (oligopeptide or polypeptide) containing a hydroxy group, suchas serine, threonine and tyrosine, or peptides containirg them, or aderivative of said amino acid or peptide selected from esters such asalkyl, preferably methyl, esters, and N-protected derivatives whereinthe N-protecting group is for example tert-butyloxy, carbobenzoxy ortrityl, and said hydroxylated amino acid or peptide or derivativethereof is linked to the COO⁻ group of the BChl derivative through itshydroxy group. Examples of such amino acid derivatives are serine methylester, N-tert-butyloxycarbonyl-serine, N-trityl-serine methyl ester,tyrosine methyl ester, and N-tert-butoxy-tyrosine methyl ester, and anexample of such a peptide is N-carbobenzoxy-seryl serine methyl ester,all of them prepared as described in the above-mentioned EP 0584552.

In another embodiment, R₅, R₆, R₇ and/or R′₇ is the residue of an aminoacid or peptide (oligo or polypeptide) linked to —CO group through anamide bond (Y is NH).

In a further embodiment, R₅, R₆, R₇ or R′₇ is the residue of acell-specific or tissue-specific ligand selected from peptides andproteins, which are exemplified by, but not limited to, hormonepeptides, for example, melanocyte-stimulating hormones, e.g. α-MSH, andantibodies, e.g. immunoglobulins, and tumor-specific antibodies. Thepeptide or protein may be linked directly to the CO group via an ester,thioester or amide bond, or it may be linked via an ester or amide bondto an end functional group of the C₁-C₂₅ hydrocarbyl radical selectedfrom OH, COOH and NH₂.

As described in the above-mentioned EP 0584552, by conjugation of Bchlwith different amino acids, and further conjugation of the Bchl aminoacid conjugates with hormones, growth factors or derivatives thereof, ortumor-specific antibodies, or any other cell-specific ligands, suitablesite-directed photosensitizers are obtained.

In one embodiment, the negatively charged group COO⁻, COS⁻, SO₃ ⁻, orPO₃ ²⁻ according to the invention originates from the functional groupCOOH, COSH, SO₃H, or PO₃H₂, respectively, of substituted hydrocarbylchains of R₅, R₆, R₇ and/or R′₇. In another embodiment, the COOH, COSH,COO⁻, and/or COS⁻ group is derived from R₁, R₂, and R₄ being OH or SH,O⁻R₈ ⁺ or S⁻R₈ ⁺, respectively, i.e., when a carboxylic orthiocarboxylic group or a carboxylate or thiocarboxylate anion ispresent at the position 13¹, 15¹ (m is 0), 15² (m is 1), and/or 17³.

The cation R₈ ⁺ may be a monovalent or divalent cation derived from analkaline or alkaline earth metal such as K⁺, Na⁺, Li⁺, NH₄ ⁺, Ca⁺, morepreferably K⁺; or R₈ ⁺ is a cation derived from an amine.

In one preferred embodiment, the bacteriochlorophyll derivative of theinvention has the formula I wherein:

M represents divalent Pd;

R₁ is —NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺, —NH—(CH₂)_(n)—COO⁻R₈ ⁺; —NH—(CH₂)_(n)—PO₃²⁻ (R₈ ⁺)₂;

R₂ is methoxy; and

R₃ is —C(═O)—CH₃;

R₈ ⁺ is a monovalent cation such as K⁺, Na⁺, Li⁺, NH₄ ⁺; and

n is an integer from 1 to 10, preferably 2 or 3.

According to this embodiment, in the compound of formula I, R₁ ispreferably a group —NH—(CH₂)_(n)—SO₃R₈ ⁺, wherein n is 3 and R₈ ⁺ is K⁺.

In another preferred embodiment, the bacteriochlorophyll derivative ofthe invention has the formula II wherein:

M represents 2H, divalent Pd, Cu, or Zn or trivalent Mn;

R₁ is —O⁻R₈ ⁺, —NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺, —NH—(CH₂)_(n)—COO⁻R₈ ⁺;—NH—(CH₂)_(n)—PO₃ ²⁻ (R₈ ⁺)₂; or Y—R₅ wherein Y is O, S or NH and R₅ isthe residue of an amino acid, a peptide or a protein;

R₂ is C₁-C₆ alkoxy such as methoxy, ethoxy, propoxy, butoxy, morepreferably methoxy;

R₃ is —C(═O)—CH₃, —CH═N—(CH₂)_(n)—SO₃ ⁻R₈ ⁺; —CH═N—(CH₂)_(n)—COO⁻R₈ ⁺;—CH═N—(CH₂)_(n)— PO₃ ²⁻ (R₈ ⁺)₂; —CH₂—NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺;—NH—(CH₂)_(n)—COO⁻R₈ ⁺; or —NH—(CH₂)_(n)—PO₃ ²⁻(R₈ ⁺)₂;

R₄ is —NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺; —NH—(CH₂)_(n)—COO⁻R₈ ⁺; —NH—(CH₂)_(n)—PO₃²⁻(R₈ ⁺)₂;

R₈ ⁺ is a monovalent cation such as K⁺, Na⁺, Li⁺, NH₄ ⁺, more preferablyK⁺;

m is 1, and n is an integer from 1 to 10, preferably 2 or 3.

In a more preferred embodiment of the invention, the bacteriochlorophyllderivative has the formula II and M is Pd.

In another more preferred embodiment, the invention relates to abacteriochlorophyll derivative of the formula II wherein:

M is Pd;

R₁ is —O⁻R₈ ⁺, —NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺, or Y—R₅ wherein Y is O, S or —NHand R₅ is the residue of a protein, preferably immunoglobulin;

R₂ is C₁-C₆ alkoxy such as methoxy, ethoxy, propoxy, butoxy, morepreferably methoxy;

R₃ is —C(═O)—CH₃, —CH═N—(CH₂)_(n)—SO₃ ⁻ R₈ ⁺; or CH₂—NH—(CH₂)_(n)—SO₃⁻R₈ ⁺;

R₄ is —NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺; NH—(CH₂)_(n)—COO⁻R₈ ⁺; NH—(CH₂)_(n)—PO₃²⁻(R₈ ⁺)₂;

R₈ ⁺ is a monovalent cation such as K⁺, Na⁺, Li⁺, NH₄ ⁺, more preferablyK⁺;

and m is 1, and n is 2 or 3, more preferably 2.

An example of a bacteriochlorophyll derivative of the invention having asole negatively charged group (SO₃ ⁻) at position 17 is represented bythe compound of Formula I identified in the List of Compoundshereinafter as compound 7.

Examples of bacteriochlorophyll derivatives of the invention having twonegatively charged groups at positions 13 and 17 include the compoundsof Formula II identified in the List of Compounds hereinafter ascompounds 4, 8, 8, 10, 11, 12, 13, 14, 15. In a most preferredembodiment, the compound of the invention is compound 4.

Examples of bacteriochlorophyll derivatives of the invention havingthree negatively charged groups at positions 3, 13 and 17 include thecompounds of formula II identified in the List of Compounds hereinafteras compounds 9 and 16. The compound 13 has one negatively charged groupat position 13 and a —COOH group as part of the protein molecule atposition 17³, and the compound 15 has one divalent negatively chargedgroup at position 13 and a —COO⁻ group at position 17³.

The compounds of the invention can be prepared, for example, by themethods as depicted in Scheme 1 herein. For the preparation of compoundswherein R₅ is the residue of an amino acid, peptide or protein, themethods described in the above-mentioned EP 0584552, particularly thecatalytic condensation method, can be used as shown in Scheme 1 for thereaction with the aminosulfonic acids taurine and homotaurine.

Thus, a method for the preparation of compounds of formula II wherein R₁is —O⁻R₈ ⁺; R₂ is —OCH₃; R₃ is acetyl; R₄ is a group —NH—(CH₂)_(n)—SO₃⁻R₈ ⁺; R₈ ⁺ is a monovalent cation; m is 1 and n is 1 to 10, comprises:(i) reacting the corresponding M-bacteriopheophorbide of formula Iwherein R₁ is OH with an aminosulfonic acid of the formulaH₂N—(CH₂)_(n)—SO₃H in a R₈ ⁺-buffer; and (ii) isolating the desiredcompound of formula II.

For preparation of the compound 4, the method comprises reactingPd-bacteriopheophorbide a 3 with taurine of the formula H₂N—(CH₂)₂—SO₃Hin a K⁺-buffer, and isolating the desired compound.

For preparation of the compound 5, the method comprises reactingbacteriopheophorbide a 2 with taurine of the formula H₂N—(CH₂)₂—SO₃H ina K⁺-buffer, and isolating the desired compound.

For preparation of the Cu and Zn compounds 10, 11, the method comprisesdirect insertion of the central metal Cu or Zn atom by reacting thecompound 5 with copper acetate or zinc acetate, respectively, while forpreparation of the Mn compound 12, insertion of the central metal Mnatom is carried out by transmetalation by first reacting the compound 5with cadmium acetate and then with manganese chloride.

A method for the preparation of compounds of formula II wherein R₁ is—O⁻R₈ ⁺; R₂ is —OCH₃; R₃ is acetyl; R₄ is a group —NH—(CH₂)_(n)—COO⁻R₈⁺; R₈ ⁺ is a monovalent cation; m is 1 and n is 1 to 10, comprises: (i)reacting the corresponding M-bacteriopheophorbide of formula I whereinR₁ is OH with an aminocarboxylic acid of the formula H₂N—(CH₂)_(n)—COOHin a R₈ ⁺-buffer; and (ii) isolating the desired compound of formula II.

Thus, for preparation of the compound 14, the method comprises reactingPd-bacteriopheophorbide a 3 with β-alanine of the formulaH₂N—(CH₂)₂—COOH in a K⁺-buffer, and isolating the desired compound.

A method for the preparation of compounds of formula II wherein R₁ is—O⁻ R₈ ⁺; R₂ is —OCH₃; R₃ is acetyl; R₄ is a group —NH—(CH₂)_(n)—PO₃ ²⁻(R₈ ⁻)₂; R₈ ⁺ is a monovalent cation; m is 1 and n is 1 to 10,comprises: (i) reacting the corresponding M-bacterio-pheophorbide offormula I wherein R₁ is OH with an aminophosphonic acid of the formulaH₂N—(CH₂)_(n)—PO₃H₂ in a R₈-buffer; and (ii) isolating the desiredcompound of formula II.

Thus, for preparation of the compound 15 the method comprises reactingPd-bacteriopheophorbide a 3 with 3-amino-1-propanephosphonic acid of theformula H₂N—(CH₂)₃—PO₃H₂ in a K⁺-buffer, and isolating the desiredcompound. For the preparation of compounds having the same negativelycharged groups at positions 13 and 17, the correspondingM-bacteriopheophorbide can be reacted with an excess of the reagent suchas aminosulfonic, aminocarboxylic or aminophosphonic acid as describedabove, and isolation of the desired 13,17-disubstituted derivative offormula II, or a different route can be followed as depicted in Scheme 1herein and described below.

Thus, a method for the preparation of compounds of formula II wherein R₁and R₄ are each a group —NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺; R₂ is —OCH₃; R₃ isacetyl; R₈ ⁺ is a Monovalent cation; m is 1 and n is 1 to 10, comprises:(i) coupling the corresponding M-bacteriopheophorbide of formula Iwherein R₁ is OH with N-hydroxy-sulfosuccinimide (sulfo NHS) in thepresence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC); (ii)reacting the resultingM-bacteriopheophorbide-17³-N-hydroxysulfosuccinimide ester with anexcess of an aminosulfonic acid of the formula H₂N—(CH₂)_(n)—SO₃H in aR₈ ⁺-buffer, thus obtaining a compound of formula I having a solenegatively charged group at position 17; (iii) reacting this productwith an excess of H₂N—(CH₂)_(n)—SO₃H in a R₈ ⁺-buffer; and isolating thedesired compound of formula II.

For the preparation of the compound §, the reaction is carried out withan excess of homotaurine of the formula H₂N—(CH₂)₃—SO₃H.

When the aminosulfonic acid is replaced by aminocarboxylic oraminophosphonic acid, the corresponding carboxylate and phosphonatederivatives are obtained.

The compounds of the invention, also referred herein sometimes by theterm “pigments”, present sufficient high polarity to be water solubleand injected in aqueous solutions with no added surfactants. In oneembodiment, for the preferred sulfonated-Pd-Bchl compound 4, alsobiodistribution and pharmacokinetics are shown and, based thereon, it isassumed that this and the other derivatives of the invention remain inthe circulation, and for a very short time. Therefore they are goodsensitizers for vascular-targeting PDT. Treatment of M2R melanoticmelanoma and HT-29 human colon carcinoma xenografts in mice shownherein, demonstrate the selective effect of the pigment on the tumorvasculature. The suggested protocol with sulfonated-Pd-Bchl 4 consideredthe short clearance time of the drug. On the ground of their selectiveeffect on the tumor vasculature, these compounds can be used for tumoras well as age-related macular degeneration and other tissuesabnormalities that depend on neovascularization.

Thus, in another aspect, the present invention provides a pharmaceuticalcomposition comprising a bacteriochlorophyll derivative of the inventionand a pharmaceutically acceptable carrier.

In a preferred embodiment, the pharmaceutical composition comprises abacteriochlorophyll derivative of formula I or II herein, morepreferably a sulfonated derivative of formula II, most preferably thecompound 4.

The anionic bacteriochlorophyll derivatives of the present invention areformulated into final pharmaceutical compositions for administration tothe patient or applied to an in vitro target using techniques well-knownin the art, for example, as summarized in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Penna., latest edition. Thecompositions can be administered systemically, in particular byinjection, or can be used topically.

The anionic Bchl compounds of the invention have similar opticalabsorption and photophysical characteristics as the respectivenon-anionic Bchls and, therefore, once residing within the treatedtissue, they are expected to be efficient photodynamic agents. They canthus be useful as photosensitizers as therapeutic and diagnostic agents,for example for treatment of several cancer types such as, but notlimited to, melanoma, prostate, brain, colon, ovarian, breast, skin,lung, esophagus and bladder cancers and other hormone-sensitive tumors,as well as for treatment of age-related macular degeneration, and forkilling cells, viruses, fungi and bacteria in samples and living tissuesas well known in the art of PDT and other photosensitizer applications.

The new water-soluble Bchl derivatives of the invention are useful, forexample, in sensitizing neoplastic cells or other abnormal tissue todestruction by irradiation either in vivo or ex vivo using light ofappropriate wavelength. It is believed that the energy ofphotoactivation is transferred to endogenous oxygen to convert it tosinglet oxygen, and/or other reactive oxygen species (ROS) such assuperoxide and hydroxyl radicals, which are considered to be responsiblefor the cytotoxic effect. In addition, the photoactivated forms of theBchls fluoresce, which fluorescence can aid in localizing tumors orother sites to which the Bchl derivative is administered.

Examples of indications, known in the art, that can be treated with thebacteriochlorophyll derivatives of the present invention, includedestruction of tumor tissue in solid tumors and dissolution of plaquesin blood vessels (see, e.g., U.S. Pat. No. 4,512,762). Particularly,these derivatives are suitable for vascular-targeted PDT because oftheir minimal retention in the circulation and because they are taken-uponly minimally by non-circulating tissues such as skin and muscle. Thus,these compounds enable reactive oxygen species (ROS) generation limitedto the interior vessels upon excitation and, thereby, cause selectiveresponse of abnormal vessels such as those present in tumors andage-related macular degeneration. In addition, the bacteriochlorophyllderivatives are useful for selective destruction in treatment of topicalconditions such as acne, athlete's foot, warts, papilloma, andpsoriasis, for treatment of benign prostate hypertrophy and forsterilization of biological products such as blood for transfusion, bydestruction of infectious agents.

The pharmaceutical compositions of the invention will be administered tothe patient by standard procedures used in PDT. The amount of theanionic Bchl derivative of the invention to be administered to anindividual in need and the route of administration will be establishedaccording to the experience accumulated with other porphyrins used inPDT, and will vary depending on the choice of the derivative used asactive ingredient, the condition, e.g. the kind of tumor, to be treated,the stage of the disease, age and health conditions of the patient, andthe judgement of the physician, but will be much lower than currentlyused dosage of Photofrin II of about 20-40 mg HPD/kg body weight. Thepreferable routes of administration are intravenous or direct injectioninto the solid tumor of the aqueous solution of the active compoundcomprising conventional pharmaceutically acceptable carriers andadditives, and topical treatment of skin tumors with suitable topicalcompositions.

The wavelength of irradiating light is preferably chosen to match themaximum absorbance of the bacteriochlorophyll photosensitizer. Thesuitable wavelength for any of the compounds can readily be determinedfrom its absorption spectrum.

In addition to in vivo use, the anionic Bchl derivatives of theinvention can be used in the treatment of materials in vitro to killharmful viruses or infectious agents, such as harmful bacteria. Forexample, blood and blood plasma to be used for future transfusion can betreated with a Bchl of the invention and irradiated to effectsterilization.

The conjugation of proteins, e.g., hormones, growth factors or theirderivatives, antibodies, peptides that bind specifically to target cellsreceptors, and of cell nutrients, e.g. tyrosine, to the Bchl moiety ismeant to increase their retention in tumor and treated sites. Increasingthe red shift allows for a greater depth of penetration, while keepingthe ubiquity of the natural system. Replacement of the Mg by othermetals is meant to optimize the intrinsic and metabolic stability of theBchl moiety and its intersystem crossing to the excited triplet state,and also opens the possibility for new diagnostic procedures.

Tumor-specific antibodies and peptides that have high affinity toneoendothelial cells will preferentially target the Bchl moieties to thetumor or treated site, while hormones and cell nutrients may also betaken up by the normal non-transformed counterparts. However, the cellsselected as targets to hormones and cell nutrients, such as melanocytesand neoendothelial cells are scattered among other cells under normalconditions and when transformed into malignant cells, cluster into solidtumors. As a result, the concentration of the photosensitizer in thevascular and/or cellular compartments of the malignant tissue isexpected to increase dramatically relative to its concentration in thenormal tissue, where cells are more dispersed, assuring amplification ofthe PDT effect in the tumor site. This enables effective use of lightdoses, lower than the damaging threshold of the normal tissue, thusreducing the need for spatially well-defined irradiation. In addition,having very strong fluorescence, the site-directed Bchl can be used forfluorescence labeling of the tumor site(s) or other targets.

In one most preferred embodiment of the present invention, the targetfor treatment with the sensitizers of the invention are abnormal bloodvessels, particularly blood vessels of solid tumors and age-relatedmacular degeneration, due to the inherent difference of sensitivity ofnormal and abnormal blood vessels to the suggested PDT protocolsdescribed herein.

The invention further relates to a method of photodynamic therapy, whichcomprises administering to an individual in need an effective amount ofa Bchl derivative of the invention, followed by local irradiation.

In one embodiment, the PDT method of the invention is used for treatmentof cancer and comprises administering to a patient afflicted with asolid tumor cancer a therapeutically effective amount of a Bchlderivative according to the invention, and then irradiating the tumorsite with strong light sources at 670-780 nm.

The Bchl derivatives of the invention are also useful forphotodestruction of normal or malignant animal cells as well as ofmicroorganisms in culture, enabling selective photodestruction ofcertain types of cells in culture or infective agents; for targeting ofthe porphyrin moiety to selected cells by attachment to specificpolypeptides, such as hormones or other receptor ligands, to cell- ortissue-specific antibodies or to other ligands, e.g., lectins; forfluorescent labeling/tagging of molecules for analytical purposes inlaboratory, diagnostic and industrial applications; and for fluorescentlabeling of animal cells or microorganisms or particles for laboratory,diagnostic or industrial applications. They can replace several of thecurrently used fluorescence tags, such as fluorescein isothiocyanate(FITC) or phycoerythrine, due to their superior extinction coefficientsand higher fluorescence yield.

For diagnostic purposes, the Bchl derivatives of the invention may beused alone or may be labeled with a radioisotope or other detectingmeans as known in the art. For example, the Bchl derivative can beradioactively-labeled by standard procedures, e.g., with ⁶⁷Ga, ¹¹¹In,²⁰¹Tl, ⁹⁹mTc, and the radioactive diagnostic agent is administered tothe patient, preferably by intravenous injection. After some hours, thelocus of the cancer may be imaged by standard procedures.

The invention further provides the use of the Bchl derivatives of theinvention for ex-vivo or in vitro killing of cells or infectious agentssuch as bacteria, viruses, parasites and fungi in a biological product,e.g. blood, which comprises treating the infected sample with thecompound of the invention followed by illumination of the sample.

The invention will now be illustrated by the following non-limitativeExamples.

EXAMPLES

For convenience and better understanding, the section of the Examples isdivided into two subsections: (I) the Chemical Section, describing thesynthesis of the water-soluble derivatives and intermediates 4-16, and(II) the Biological Section, describing the biological activity of thenew Bchl derivatives.

I Chemical Section

In the Examples herein, the derivatives of the invention (4-5, 7-9, and10-16) and the intermediates (1-3, and 6) will be presented by theirrespective Arabic numbers in bold and underlined according to thefollowing List of Compounds. The corresponding formulas appear in theScheme at the end of the specification, just before the claims.

LIST OF COMPOUNDS

-   1. Bacteriochlorophyll a (Bchl a)-   2. Bacteriopheophorbide a (Bpheid a)-   3. Pd-Bacteriopheophorbide a (Pd-Bpheid a)-   4. Palladium 3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin    13¹-(2-sulfo-ethyl)amide dipotassium salt [Example 1]-   5. 3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin    13¹-(2-sulfoethyl)amide dipotassium salt [Example 2]-   6. Palladium bacteriopheophorbide a 17³-(3-sulfo-1-oxysuccinimide)    ester sodium salt [Example 6]-   7. Palladium Bacteriopheophorbide a 17³-(3-sulfopropyl)amide    potassium salt [Example 7]-   8. Palladium 3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin    13¹,17³-di(3-sulfopropyl)amide dipotassium salt [Example 8]-   9. Palladium    3¹-(3-sulfopropylimino)-15-methoxycarbonylmethyl-Rhodobacteriochlorin    13¹,17³-di(3-sulfopropyl)amide tripotassium salt [Example 9]-   10. Copper(II) 3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin    13¹-(2-sulfoethyl)amide dipotassium salt [Example 3]-   11. Zinc 3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin    13¹-(2-sulfoethyl) amide dipotassium salt [Example 4]-   12. Manganese(III)    3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin    13¹-(2-sulfoethyl)amide dipotassium salt [Example 5]-   13. Palladium 3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin    13¹-(2-sulfoethyl)amide, 17³-(N-immunoglobulin G)amide potassium    salt] [Example 10]-   14. Palladium 3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin    13¹-(2-carboxyethyl)amide dipotassium salt [Example 11]-   15. Palladium 3¹-oxo-15-methoxycarbonyhnethyl-Rhodobacteriochlorin    13¹-(3-phosphopropyl)amide tripotassium salt [Example 12]-   16. Palladium    3¹-(3-sulfopropylamino)-15-methoxycarbonylmethyl-Rhodobacteriochlorin    13¹,17³-di(3-sulfopropyl)amide tripotassium salt [Example 13]

Materials and Methods

(i) Bchl a (1) was extracted and purified from lyophilized cells ofRhodovolum Sulfidophilum as previously described (WO 00/33833).

(ii) Palladium bacteriopheophorbide (Pd-Bpheid, a) was either preparedas previously described (WO 00/33833) or it was obtained from StebaBiotech Ltd. through Negma-Lerads, France.

(iii)3-Amino-1-propane sulfonic acid (homotaurine) and 3-amino-1-propanephosphonic acid were purchased from Aldrich (USA), and 2-aminoethanesulfonic acid (taurine) and 3-aminopropionic acid (β-alanine) werepurchased from Sigma (USA), N-hydroxy-sulfosuccinimide (sulfo-NHS) waspurchased from Pierce (USA),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was purchased fromFluka (Switzerland).

(iv) Chemicals and solvents of analytical grade were generally usedexcept when performing HPLC, where HPLC-grade solvents were applied.

(v) TLC: silica plates (Kieselgel-60, Merck, Germany);chloroform-methanol (4:1, v/v).

(vi) ¹H Nuclear magnetic resonance (NMR) spectra were recorded on AvanceDPX 250 instrument (Broker, France) and reported in ppm (δ) downfieldfrom tetramethylsilane with residual solvent peaks as the internalstandards.

(vii) The extinction coefficients of the Pd-derivatives were determinedby correlating the Pd concentration (using flame photometry with PdCl₂as a standard) with the optical density of the examined solution at theparticular wavelength.

(viii) Electrospray ionization mass spectra (ESI-MS) were recorded on aplatform LCZ spectrometer (Micromass, England).

(ix) Inductively-Coupled Plasma Mass Spectrometry (ICP-MS) was performedfor determination of Pd concentrations using an ELAN-6000 instrument(Perkin Elmer, Conn.).

(x) Optical absorption of the different complexes was recorded withGenesis-2 (Milton Roy, England) and V-570 (JASCO, Japan)spectrophotometers.

(xi) HPLC was performed using an LC-900 instrument (JASCO, Japan)equipped with a UV-915 diode-array detector.

CHEMICAL EXAMPLES Example 1. Palladium3¹-oxo-15-methoxyearbonylmethyl-Rhodobacteriochlorin13¹-(2-sulfoethyl)amide dipotassium salt (Compound 4)

Nine hundred and thirty five (935) mg of Pd-Bpheid (3) were dissolved ina 1 L round bottom flask with 120 ml of DMSO while stirring under Argon(bubbled in the solution). Taurine (1288 mg) was dissolved in 40 ml of1M K₂HPO₄ buffer, and the pH of the solution was adjusted to 8.2 (withHCl). This aqueous solution was added into the DMSO solution whilestirring, and the Argon was bubbled in the solution for another 20minutes. Then the reaction mixture was evaporated at 30° C. for 3.5hours under 2 mbar and then for another 2 hours at 37° C. to a completedryness. The dry solids were dissolved in 300 ml of MeOH and the coloredsolution was filtered through cotton wool to get rid of buffer salts andtaurine excess.

The progress of the reaction was determined by TLC (R_(f) of unreactedPd-Bpheid is 0.8-0.85 and of the reaction (aminolysis) product is0.08-0.1) and by following the optical absorption spectrum of thereaction mixture after liophylimtion and resolubilization in MeOH. Theabsorption spectrum was characterized by a Q_(y) transition shift from756 nm (for Pd-Bpheid) to 747 nm (for the product 4) and by Q_(x) shiftfrom 534 nm of Pd-Bpheid to 519 nm (of the product 4). The MeOH wasevaporated and the product 4 was purified by HPLC with ODS-A 250×20 S10Pμm column (YMC, Japan). Solvent A: 95% 0.005 M phosphate buffer, pH 8.0and 5% MeOH. Solvent B: 100% MeOH. The dry solid was dissolved in 42 mlof distilled water and injected in portions of 1.5 ml each.

The elution profile is described in Table 1. The product 4 (Scheme 1,see below) was eluted and collected at ˜9-11 minutes. The mainimpurities, collected after at 4-7 min (ca 5-10%), corresponded tobyproduct(s) with the proposed structure 7. Peaks at 22-25 min (ca 2-5%)possibly corresponded to the iso-form of the main product 4 anduntreated Pd-Bpheid residues.

TABLE 1 Gradient profile of purification of compound 4 Time (min) Flow(ml/min) A % B % 0 12 55 45 14 12 30 70 14.1 6 30 70 16 6 0 100 18 6 0100 24 6 55 45 29 6 55 45 30 0.5 55 45

The solvent (aqueous methanol) was evaporated under reduced pressure.Then, the purified product 4] was re-dissolved in ˜150 ml MeOH andfiltered through cotton wool. The solvent was evaporated again and thesolid pigment 4 was stored under Ar in the dark at −20° C. The reactionyield: ˜90% (by weight, relative to 3).

The structure of product 4 was confirmed by electrospray massspectroscopy. (ESI-MS, negative mode, FIG. 2), (peaks at 875 (M⁻—K—H),859 (M⁻-2K—H+Na), 837 (M⁻-2K), 805 (M2K—H-OMe), 719) and ¹H-NMR spectrum(FIG. 4 in MeOH-d₄). Table 4 provides the shifts (in ppm units) of themajor NMR peaks.

Optical absorption (UV-VIS) spectrum (MeOH): λ, 747 (1.00), 516 (0.13),384 (0.41), 330 (0.50); ε₇₄₇ (MeOH) is 1.2×10⁵ mol⁻¹ cm⁻¹.

NMR (MeOH-d₄): 9.38 (5-H, s), 8.78 (10-H, s), 8.59 (20-H, s), 5.31 and4.95 (15¹-CH₂, dd), 4.2-4.4 (7,8,17,18-H, m), 3.88 (15³-Me, s), 3.52(2¹-Me, s), 3.19 (12¹-Me, s), 3.09 (3²-Me, s), 1.92-2.41, 1.60-1.75(17¹, 17²-CH₂, m), 2.19 (8¹-CH₂, m), 1.93 (7¹-Me, d), 1.61 (18¹-Me, d),1.09 (8²-Me, t), 3.62, 3.05 (CH₂'s of taurine). Octanol/water partitionratio is 40:60.

Example 2. Preparation of3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹-(2-sulfoethyl)amide dipotassium salt (Compound 5)

One hundred and sixty (160) mg of taurine were dissolved in 5 ml of 1MK₂HPO₄ buffer, and the pH of the solution was adjusted to 8.2. Thissolution was added to 120 mg of compound 2 dissolved in 15 ml of DMSO,and the reaction and following purification were analogous to thosedescribed in previous Example. Absorption spectrum (MeOH): λ, 750(1.00), 519 (0.30), 354 (1.18) nm.

ESI-MS (−): 734 (M⁻-2K).

NMR (MeOH-d₄): 9.31 (5-H, s), 8.88 (10-H, s), 8.69 (20-H, s), 5.45 and5.25 (15¹-CH₂, dd), 4.35 (7,18-H, m), 4.06 (8,17-H, m), 4.20 and 3.61(2-CH₂, m of taurine), 3.83 (15³-Me, s), 3.63 (2¹-Me, s), 3.52 (3-CH₂, mof taurine), 3.33 (12¹-Me, s), 3.23 (3²-Me, s), 2.47 and 2.16 (17¹-CH₂,m), 2.32 and 2.16 (8¹-CH₂, m), 2.12 and 1.65 (17²-CH₂, m), 1.91 (7¹-Me,d), 1.66 (18¹-Me, d), 1.07 (8²-Me, t).

Octanol/water partition ratio is 60:40.

Example 3. Preparation of copper(II)3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹-(2-sulfoethyl)amide dipotassium salt (Compound 10)

Fifty (50) mg of compound 5 of Example 2 and 35 mg of copper (II)acetate were dissolved in 40 ml of methanol, and argon was bubbled intosolution for 10 minutes. Then 500 mg of palmitoyl ascorbate was added,and the solution was stirred for 30 min. The absorption spectrum wascharacterized by a Q_(y) transition shift from 750 nm (for 5) to 768 nm(for the product 10) and by Q_(x) shift from 519 nm of 5 to 537 nm (ofthe product 10). Then the reaction mixture was evaporated, re-dissolvedin acetone and filtered through cotton wool to get rid of acetate saltexcess. The acetone was evaporated and the product was additionallypurified by HPLC at the conditions mentioned above with the elutionprofile, described in Table 2.

The solvent (aqueous methanol) was evaporated under reduced pressure.Then, the purified pigment 10 was re-dissolved in methanol and filteredthrough cotton wool. The solvent was evaporated again and the solidpigment 10 was stored under Ar in the dark at −20° C. Reaction yield:˜90%.

TABLE 2 Gradient profile of purification of compound 10 Time (min) Flow(ml/min) A % B % 0 12 58 42 14 12 45 55 14.1 6 45 55 16 6 0 100 18 6 0100 24 6 58 42 29 6 58 42 30 0.5 58 42

Absorption spectrum (MeOH): λ, 768 (1.00), 537 (0.22), 387 (0.71) and342 (0.79) nm.

ESI-MS (−): 795 (M⁻-2K).

Octanol/water partition ratio is 40:60.

Example 4. Preparation of zinc3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹-(2-sulfoethyl)amide dipotassium salt (Compound 11)

Zn insertion into compound 5 was carried out with Zn acetate in aceticacid as previously described (U.S. Pat. No. 5,726,169). Finalpurification was carried out by HPLC in the same conditions as forcompound 5 in Example 2 above.

Absorption spectrum (MeOH): λ, 762 (1.00), 558 (0.26), 390 (0.62) and355 (0.84) nm.

Octanol/water partition ratio is 50:50.

Example 5. Preparation of manganese(III)3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹-(2-sulfoethyl)amide dipotassium salt (Compound 12)

Mn insertion into compound 5 was carried out with Zn acetate in aceticacid as previously described (WO 97/19081; U.S. Pat. No. 6,333,319) withsome modifications. Thus, fifty (50) mg of compound 5 in 10 ml of DMFwere stirred with 220 mg of cadmium acetate and heated under argonatmosphere at 110° C. about 15 min (Cd-complex formation is monitored byshifting Q_(x) transition absorption band from 519 to 585 nm inacetone). Then the reaction mixture was cooled and evaporated. The dryresidue was re-dissolved in 15 ml of acetone and stirred with manganese(II) chloride to form the Mn(III)-product 12. The product formation ismonitored by shifting Q_(x) transition band from 585 to 600 nm and Q_(y)transition band from 768 to 828 nm in acetone. The acetone wasevaporated and the product 12 was additionally purified by HPLC in theconditions mentioned in Example 2 above with the elution profiledescribed in Table 3 below where thelsolvent system consists of: A-5%aqueous methanol, B-methanol.

TABLE 3 Gradient profile of purification of compound 12 Time (min) Flow(ml/min) A % B % 0 8 95 5 14 8 55 45 14.1 8 55 45 16 8 0 100 18 8 0 10024 8 95 5 29 8 95 5 30 0.5 95 5

The solvent (aqueous methanol) was evaporated under reduced pressure andthe solid pigment 12 was stored under Ar in the dark at −20° C.

Absorption spectrum (MeOH): λ, 828 (1.00), 588 (0.32) and 372 (0.80) nm.Octanol/water partition ratio is 5:95.

Example 6. Preparation of palladium bacteriopheophorbide a17³-(3-sulfo-1-oxy-succinimide)ester sodium salt (Compound 6)

Fifty (50) mg of Pd-Bpheid (compound 2), 80 mg ofN-hydroxy-sulfosuccinimide (sulfoNHS) and 65 mg of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were mixed in 7 mlof dry DMSO for overnight at room temperature. Then the solvent wasevacuated under reduced pressure. The dry residue was re-dissolved inchloroform (ca. 50 ml), filtered from insoluble material, andevaporated. The conversion was ab. 95% (TLC). The product 6 was usedlater on without further chromatographic purification. ESI-MS (−): 890(M⁻-Na).

NMR (CDCl₃): 9.19 (5-H, s), 8.49 (10-H, s), 8.46 (20-H, s), 5.82 (13²-H,s), 4.04-4.38 (7,8,17,18-H, m), 3.85 (13⁴-Me, s), 3.47 (2¹-Me, s), 3.37(12¹-Me, s), 3.09 (3²-Me, s), 1.77 (7¹-Me, d), 1.70 (18¹-Me, d), 1.10(8²-Me, t), 4.05 (CH₂ of sNHS), 3.45 (CH of s NHS).

Example 7. Preparation of palladium bacteriopheophorbide a17³-(3-sulfopropyl) amide potassium salt (Compound 7)

Ten (10) mg of compound 6 in 1 ml of DMSO was mixed with 20 mg ofhomotaurine (3-amino-1-propane-sulfonic acid) in 1 ml of 0.1 MK-phosphate buffer, pH 8.0 for overnight. Then the reaction mixture waspartitioned in chloroform/water. The organic layer was dried overanhydrous sodium sulfate and evaporated. The dry residue wasre-dissolved in chloroform-methanol (19:1) and applied to achromatographic column with silica. The product 7 was obtained withchloroform-methanol (4:1) elution. The yield was about 80-90%.

ESI-MS (−): 834 (M-K) m/z.

NMR (MeOH-d₄): 9.16 (5-H, s), 8.71 (10-H, s), 8.60 (20-H, s), 6.05(13²-H, s), 4.51, 4.39, 4.11, 3.98 (7,8,17,18-H, all m), 3.92 (13⁴-Me,s), 3.48 (2¹-Me, s), 3.36 (12¹-Me, s), 3.09 (3²-Me, s), 2.02-2.42 (17¹and 17²-CH₂, m), 2.15 (8¹-CH₂, q), 1.81 (7¹-Me, d), 1.72 (18¹-Me, d),1.05 (8²-Me, t), 3.04, 2.68, and 2.32 (CH₂'s of homotaurine, m).

Example 8. Preparation of palladium3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹,17³-di(3-sulfopropyl)amide dipotassium salt (Compound 8)

Ten (10) mg of compound 6 or 7 were dissolved in 3 ml of DMSO, mixedwith 100 mg of homotaurine in 1 ml of 0.5 M K-phosphate buffer, pH 8.2,and incubated overnight at room temperature. The solvent was thenevacuated under reduced pressure as described above, and the product 8was purified on HPLC. Yield: 83%.

Absorption spectrum (MeOH): 747 (1.00), 516 (0.13), 384 (0.41), 330(0.50), ε₇₄₇=1.3×10⁵ mol⁻¹ cm⁻¹.

ESI-MS(−):1011 (M⁻-K), 994 (M⁻-2K+Na), 972 (M⁻-2K), 775(M⁻-2K—CO₂Me-homotaurine NHCH₂CH₂CH₂SO₃), 486 ([M-2K]/2)

NMR (MeOH-d₄): 9.35 (5-H, s), 8.75 (10-H, s), 8.60 (20-H, s), 5.28 and4.98 (15¹-CH₂, dd), 4.38, 4.32, 4.22, 4.15 (7,8,17,18-H, all m), 3.85(15-Me, s), 3.51 (2¹-Me, s), 3.18 (12¹-Me, s), 3.10 (3²-Me, s 2.12-2.41(17¹-CH₂, m), 2.15-2.34 (8¹-CH₂, m), 1.76-2.02 (17²-CH₂, m), 1.89(7¹-Me, d), 1.61 (18¹-Me, d), 1.07 (8²-Me, t). 3.82, 3.70, 3.20, 3.10,2.78, 2.32, 1.90 (CH₂'s of homotaurine at C-13¹ and C-17³)

Example 9. Palladium3¹-(3-sulfopropylimino)-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹,17³-di(3-sulfopropyl)amide tripotassium salt (Compound 2)

Compound 9 was obtained from HPLC as a minor product during synthesis of8.

Absorption spectrum (MeOH): 729 (1.00), 502 (0.10), 380 (0.69), 328(0.57).

ESI-MS (30.4.2000): 1171 (M-K+H), 1153 (M⁻-2K—H+Na), 1131 (M-2K), 566([M-K]/2), 364 ([M-3K]/3).

NMR (MeOH-d₄): 8.71 (1H), 8.63 (1.5H), 8.23 (0.5H) (5-, 10- and 20-H,all-m), 5.30 and 4.88 (15¹-CH₂, dd), 4.43 and 4.25 (7,8,17,18-H, m),3.85 (15-Me, s), 3.31 (2¹-Me, s), 3.22 (12¹-Me, s), 3.17 (3²-Me, m),1.89-2.44 (17¹ and 17²-CH₂, m), 2.25 (8¹-CH₂, m), 1.91 (7¹-Me, s), 1.64(18¹-Me, s), 1.08 (8²-Me, t), 4.12, 3.56, 3.22, 3.16, 2.80 and 2.68(CH₂'s of homotaurine).

Example 10. Palladium3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹-(2-sulfoethyl)amide, 17³-(N-immunoglobulin G)amide potassium salt(Compound 13)

Ten (10) mg of compound 4 were reacted with 20 mg of sulfo-NHS and 15 mgof EDC in 1 ml of dry DMSO for 1 hour at room temperature, then rabbitIgG (0.6 mg) in PBS (2.5 ml) was added, and the mixture was furtherincubated overnight at room temperature. The mixture was evaporated todryness, then re-dissolved in 1 ml of PBS and loaded on Sephadex G-25column equilibrated with PBS. A colored band was eluted with 4-5 ml ofPBS. The pigment/protein ratio in the obtained conjugate 13 wasdetermined by optical density at 753 and 280 nm, respectively, andvaried between 0.5/1 to 1/1 of pigment 13/protein.

Example 11. Preparation of palladium3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹-(2-carboxyethyl)amide dipotassium salt (Compound 14)

The preparation and purification of the title compound 14 were carriedout as described in Example 2, by reaction of compound 2 with3-aminopropionic acid (β-alanine) (150 mg) instead of taurine. Yield:85%.

Example 12. Preparation of palladium3¹-oxo-15-methoxycarbonylmethyl-Rhodobacteriochlorin13¹-(3-phosphopropyl)amide tripotassium salt (Compound 15)

The preparation and purification of the title compound 15 were carriedout as described in Example 2, by reaction of compound 2 with3-amino-1-propanephosphonic acid (180 mg) instead of taurine. Yield:68%.

Example 13. Palladium3¹-(3-sulfopropylamino)-15-methoxycarbonylmethyl-Rhodobacteriochimin13¹,17³-di(3-sulfopropyl)amide tripotassium salt (Compound 16)

For reduction of the inline group in 3¹-(3-sulfopropylimino) to thecorrespondent 3¹-(3-sulfopropylamino) group, compound 9 (8 mg) wasreacted by stirring with sodium cyanoborohydride (15 mg) in 5 ml ofmethanol overnight at room temperature. Then the reaction mixture wastreated with 0.05 M HCl (5 ml), neutralized with 0.01 M KOH, andevaporated. The title product 16 was purified using HPLC conditions asdescribed in Example 2. Yield: 80-90%.

II BIOLOGICAL SECTION Materials and Methods In Vitro Studies

-   -   (i) Cell Culture.

M2R mouse melanoma, H5V mouse endothelial and C6 rat glioma cells werecultured as monolayers in Dulbecco's modified Eagle's medium (DMEM)/F12containing 25 mM HEPES, pH 7.4, 10% fetal bovine serum (FBS), glutamine(2 mM), penicillin (0.06 mg/ml), and streptomycin (0.1 mg/ml)(hereinafter referred to as the “Culture Medium”). Cells were grown at37° C. in an 8% CO₂-humidified atmosphere.

(ii) Phototoxicity Assay.

To determine the photodynamic efficacy, cells were preincubated withincreasing concentrations of the pigments in the dark for the times andconditions as indicated for the individual experiments. Unboundsensitizer was removed by washing the cells once with culture medium,and the plates were illuminated at room temperature from the bottom(λ>650 nm, 12 J/cm²). The light source was a 100 W Halogen lamp (Osram,Germany) equipped with a 4-cm water filter. The cultures were placed inthe culture incubator and cell survival was determined 24 h afterillumination, by Neutral Red viability assay. Three kinds of controlswere used: (i) light control: cells illuminated in the absence ofpigments; (ii) dark control: cells treated with pigments but kept in thedark; and (iii) untreated cells that were kept in the dark

In Vivo Studies

(iii) Tumor Implantation.

M2R or C6 cells (2×10⁶) were implanted subcutaneously on the back of themice; tumors developed to the treatment size (6-8 mm) within 2-3 weeks.

(iv) Preparation of Sensitizer.

Stock solutions of the compounds of the invention were prepared prior touse by dissolving the dry pigment directly in PBS to the desiredconcentration for injection.

(v) Biodistribution and Pharmacokinetics.

Pigment 4 of the invention (6 mg/kg body) was injected to CD1 nude micevia tail vein. Mice were sacrificed at the indicated times, and samplesof the indicated organs or tissues were placed and weighed inpre-weighted vials and immediately frozen on dry ice. For examination,each sample was thawed and homogenized (1:10 w/v) in double-distilledwater. Aliquots of the homogenate (0.5 ml) were lyophilized inEppendorff test tubes. Then 0.2 ml of HNO₃ (70%, TraceSelect, Fluka) wasadded to each dry sample, incubated for 1 h at 90° C. and diluted indouble-distilled water to 10 ml. Palladium concentrations weredetermined by ICP-MS. Background was determined for each organ/tissue onidentical samples taken from untreated mice, and values were subtractedaccordingly.

(vi) PDT Protocol.

The M2R tumor-bearing mice were anesthetized and the pigment wasinjected intravenously (i.v.) via the tail vein. The tumors wereimmediately illuminated transcutaneously for 5 min by 755 mu diode laser(CeramOptec, Germany) with light dose of either 30 J/cm2 (100 mW/cm2),39 J/cm2 (130 mW/cm2) or 45 J/cm2 (150 mW/cm2). After the treatment, themice were returned to the cage. In the dark control group, the mice wereinjected i.v. with sensitizer and placed in the dark cage for 24 h. Inthe light control group, the mice were illuminated with 45 J/cm2.

(vii) Vascular Shutdown and Permeability.

Mice bearing C6 glioma tumor xenografts were treated with pigment 4 (9mg/kg) and light (100 mW/cm² for 5 min). Immediately after treatment,Evans Blue (EB; 1% in PBS) was injected (0.5 ml, i.p.). Mice werephotographed at 3 and 24 hours after treatment. The mice were sacrificed24 hours after treatment and skin flap was made for each mouse andphotographed. Then the tumor was removed with the skin above it, frozenfor 1 hour at 20° C., and then axial slice was made and the slice wasphotographed. Control mice were injected with Evans Blue at the sametime as the treated mice, and the protocol was continued as describedabove for all the mice together.

Example 14. Cytophotoxicity of the Sulfonated BacteriochlorophyllDerivatives Against Tumor Cell Cultures

The phototoxicity of compounds 4 and 8 was determined as described in(ii) above in M2R mouse melanoma and H5V mouse endothelial cells. Cellswere preincubated with increasing concentrations of the compound for 4hours, washed and illuminated or kept in the dark.

The results are shown in FIGS. 1A-1B for the bi-sulfonated compound 8 inH5V and MR2 cells, respectively, and in FIGS. 2A-2B for themono-sulfonated compound 4 (comparison) in H5V and MR2 cells,respectively. As can be seen, the phototoxicity of both pigments 4 and 8is concentration- and light-dependent, without any dark toxicity in thetested range. The LD₅₀ of both pigments is the same (˜2 μM), and issimilar in both cell lines.

The phototoxicity of the sulfonated pigments 5 and 11 was determined onM2R mouse melanoma cells. As can be seen in FIGS. 3 and 4, thephototoxicity of pigments 5 and 11 is concentration- andlight-dependent, and the LD₅₀ of both pigments is the same (˜5 μM).There is no dark toxicity within the tested range.

Example 15. Pharmacokinetics and Biodistribution of Compound 4

The first step before testing the phototoxicity of 4 toward PDT of solidmelanoma xenografts was to determine the pigment's pharmacokinetics andbiodistribution in vivo as described in section (vi) above. As can beseen in FIG. 5, about 90% of the pigment 4 cleared within the first10-min after i.v. injection with a monophasic kinetic pattern with at_(0.5) of 1.65 min (Table 4). The fast clearance of 4 from the bloodmay imply that only a small fraction (if at all) is bound to the plasmacomponents, otherwise clearance might have been slower.

TABLE 4 Pharmacokinetic parameters of 4 in mice blood. ParameterEquation y = 1.64 + 90.6e(−0.42t) T_(0.5) (min) 1.65 K_(el) (min⁻¹) 0.42Vd (ml) 2.12 CL (ml/min) 0.89 K_(el)—rate of elimination; Vd—volume ofdistribution; CL—clearance.

The biodistribution of the compound 4 shows that, in most of theexamined organs of the mouse, the pigment levels are high immediatelyafter injection and drop to almost background levels within 20-30 min,similar to their clearance rates from the blood (FIG. 6). These resultsprobably represent the pigment level in the blood trapped in the organ'svasculature as seen in spleen, lung, and heart. Furthermore, the resultsalso suggest that pigment diffusion into the organs is negligible. Thepigment 4 clears rapidly from the mouse body, and within 30 min afterinjection it is in background levels in all tissues. The clearance rateof 4 from the mouse body is much faster than Pd-Bpheid (2), whichreaches background levels only 48 hours after injection (not shown).

Example 16. Photodynamic Treatment of M2R Melanoma Xenografts in CD1Nude Mice with Sulfonated Pigment 4

Based upon the pharmacokinetic results of Example 15 above, thetreatment protocol for compound 4 was set to 5-min illuminationimmediately after pigment injection. In these experiments (see section(vii) above), a dedicated medical laser matched to the peak absorptionof 4 (CeramOptec, Germany, 755 nm) was used. In order to determine theoptimal drug/light protocol, mice were treated with drug dose of 6 mg/kgand increasing the light intensity (FIG. 7). As can be seen in theKaplan-Meier survival curve, increasing the light intensity improves themice cure rate from 43% to 60% with 30 and 45 J/cm², respectively. Whenthe drug dose was elevated to 9 mg/kg with light intensity of 30 J/cm²,there was a significant increase in the mice survival to 70% (FIG. 7).No dark toxicity was seen in animals treated with 6 or 9 mg/kg and keptin the dark.

Example 17. Selective Effect of Photodynamic Treatment with Compound 4

This experiment was carried out as described in section (vii) above.FIG. 8 illustrates the effect of photodynamic treatment on bloodperfusion in C6 xenografts implanted in mice (a, e). Treated animal thatwas administrated with Evans-Blue immediately after PDT showed edema andenhanced vascular leakage of EB into the interstitium as demonstrated bythe blue color (due to albumin-Evans Blue leakage) in the illuminatedarea when compared to the non-illuminated area in the same animal an tountreated animal (b, f). Twenty-four hours later, it can be seen that inthe treated mice, the tumor surrounding is heavily colored blue (edema;c), while the tumor remains white (no EB color) due to vascular shutdownthat occurred immediately after PDT (d). The muscle tissue under thetumor as well as the skin above and around the tumor (but within thetreated area) is blue, indicating that no vascular shutdown took place(c, d). In the untreated animal, the tumor is colored blue like othertissues (g, h). The selective enclosure of new vessels in the tumorindicates that the compounds of the invention can be used for selectivetreatment of abnormal vasculature as in age-related macular degeneration(AMD).

Example 18. PDT Treatment with Compound 4 Animal Model of AMD

Photodynamic therapy (PDT) has been developed aiming at inducinglocalized vascular occlusion of the newly formed vascular membranesemanating from the choroid (choroidal neovascularization—CNV). Inage-related macular degeneration (AMD), PDT using verteporfin reducesthe risk of visual loss secondary to CNV. The mechanism of action of PDTis thought to involve the release of reactive oxygen species whichdamage endothelial cells and activate sub endothelial clotting cascade.These events lead to the formation of thrombi within the vessel lumen.

For the treatment of choroidal neovascularization, highly selectiveparameters (Laser power density or fluence, photosensitizer dose, anddistance to light illumination (DLI)) have been developed enablingprecise focusing and targeting of the pathologic vessels and minimalsecondary damaging effects to healthy retina and choroid tissues.However, using the only photosensitizer (verteporfin) presentlyavailable for clinical use, repeated treatments are generally requiredto achieve the desired CNV occlusive effects. Thus, the danger forcollateral tissue damage is enhanced and may become a significant sideeffect of treatment.

In this experiment, we have evaluated the photodynamic treatment (PDT)potential of the hydrosoluble photosensitizer herein designated WST11 orcompound 4, and compared its characteristics to those of verteporfin.

Compound 4 is a pure and stable bacteriochlorophyll derivative isolatedas a black purple crystalline powder. It has a molecular weight of 916and is soluble in aqueous solution. It is characterized by the followingproperties: (a) 4 main absorption peaks (750, 530, 385 and 330 nm). Thestrongest absorbance of light is near the infrared (≈750 nm) wheretissue transmittance is the highest; (b) a very low cytotoxicity in thedark. Thus, tissue damage can be controlled by the light dose and lengthof exposure; (c) it is rapidly cleared from the body afteradministration. Therefore, potential skin photosensitization damage onexposure to ambient light or to the sun light is minimal; (d) generationof reactive oxygen species (ROS) is high because of efficientintersystem crossing (ISC).

The WST11 powder was diluted in endotoxin-free sterile water at aconcentration of 10 mg/ml, and shaken until complete dissolution. Thisformulation remains stable for 24 hours at 4° C. protected from light.To calculate the volume to be injected, adjustment was made according tothe rabbit weight. The appropriate volume solution was injectedintravenously as a bolus via the marginal ear vein.

The potential of Compound 4 for PDT of age-related macular degeneration(AMD) was compared to verteporfin (Visudyne®, Novartis, Switzerland)using a rabbit eye model. Pigmented rabbits (136 “Fauve de Bourgogne”rabbits, 10-12 weeks old, 2.5-3 kg; Elevage des Pins, Epeigne-sur-Dême,France) were used. Acute and long term PDT effects on the rabbit eyewere investigated for the following parameters: 1) 753 nm laser fluence(25 and 50 J/cm²), Compound 4 (also designated WST11) doses (2.5 and 5mg/kg) and distance to light illumination (DLI) of 1,5,10 and 15minutes. 2) 689 nm laser fluence (10, 50, 100 J/cm²), verteporfin doses(3, 6 and 12 mg/m²) and a constant DLI of 5 min. These PDT parametersencompassed an array of effects on the choroid and the overlying retinawere delivered for 83 seconds to induce occlusive, subthresholdocclusive and non-occlusive vascular events. Treated rabbit eyes wereexamined and followed by indirect ophthalmoscopy, fluoresceinangiography (FA) and histology at various intervals after PDT. WST11 PDTusing a fluence of 50 J/cm², 5 mg/kg drug dose and DLI of 1 minuteinduced total choroidal occlusion associated with structural lesions ofthe overlying RPE and retina in 100% of the treated eyes (FIGS. 9A-9D).Weaker, non-occlusive PDT parameters (25 J/cm², 5 mg/kg drug dose andDLI of 10 minutes) did not induce choriocapillaries occlusion norretinal lesions. Verteporfin PDT using 12 mg/m² drug dose at a fluenceof 100 J/cm² and DLI 5 minutes induced occlusive events (observed by FA)in 89% of the eyes and histology damage of the overlying retina and RPElayer in all eyes. Weaker non-occlusive verteporfin PDT parameters using3 mg/m² drug dose, fluence 10 J/cm² and DLI 5 minutes did not induce anychoriocapillaries occlusion on FA. However in these eyes, definitestructural damage of the retina and choroid tissues were observed onhistology. Similar to verteporfin, WST11 PDT induces transient occlusionof the choriocapillaries observed up to one week after treatment. UnlikeVerteporfin, WST11 PDT parameters not inducing vessel occlusion do notcause RPE or retina structural damage Thus, despite its capacity toinduce vessel obstruction, WST11 PDT does not cause damage to the RPEand overlying retina when no occlusion of the choriocapillaries takesplace. The advantages of these characteristics indicates that WST11 is asuitable candidate for PDT treatment of CNV in age-related maculardegeneration.

For the histology, enucleated eyes were dissected under a binocularmicroscope. A 4 mm biopsy punch was used to excise the full thickness oftreated zones. These tissues were fixed in glutaraldehyde, processed incacodylate buffer and embedded in plastic. Semi-thin sections wereobtained using a microtome and counter-stained with hematoxilin-eosin.These sections were analyzed using phase contrast microscopy. Specificsites of interest were further processed for TEM. Ultrathin sectionswere obtained using an ultramicrotome and counter-stained with uranylacetate.

REFERENCES

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1. A bacteriochlorophyll derivative containing at least one, preferablytwo or three, negatively charged groups and/or acidic groups that areconverted to negatively charged groups at the physiological pH,excluding pentacyclic bacteriochlorophyll derivatives having a freeCH₂CH₂COOH or a CH₂CH₂COO⁻ group at position 17, and tetracyclicbacteriochlorophyll derivatives devoid of a central metal atom andhaving a —CH₂CH₂COOH group at position 17, a —CH₂COOH or —COOH group atposition 15, a —COOH group at position 13, methyl groups at thepositions 2, 7, 12, 18, and ethyl groups at the positions 3 and
 8. 2. Abacteriochlorophyll derivative according to claim 1 containing twonegatively charged groups.
 3. A bacteriochlorophyll derivative accordingto claim 1 containing three negatively charged groups.
 4. Abacteriochlorophyll derivative according to any one of claims 1 to 3wherein said negatively charged groups are selected from the groupconsisting of COO⁻, COS⁻, SO₃ ⁻, and/or PO₃ ²⁻.
 5. A bacteriochlorophyllderivative according to claim 1 wherein said acidic groups that areconverted to negatively charged groups at the physiological pH areselected from the group consisting of COOH, COSH, SO₃H, and/or PO₃H₂. 6.A bacteriochlorophyll derivative according to any one of claims 1 to 5derived from a natural or synthetic derivative of bacteriochlorophyll,including compounds in which the central Mg atom has been deleted orreplaced by other metal atoms.
 7. A bacteriochlorophyll derivativeaccording to claim 1 of the formula I or II:

wherein M represents 2H or a metal atom selected from divalent Pd, Pt,Co, Sn, Ni, Cu, Zn and Mn, and trivalent Fe, Mn and Cr; R₁, R₂, and R₄each independently is Y—R₅; Y is O, S or NR₅R₆; R₃ is selected from—CH═CH₂, —C(═O)—CH₃, —C(═O)—H, —CH═NR₇, —C(CH₃)═NR₇, —CH₂—OR₇, —CH₂—SR₇,—CH(CH₃)—OR₇, —CH(CH₃)—SR₇, —CH(CH₃)—NR₇R′₇, —CH(CH₃)Hal, —CH₂-Hal,—CH═CR₇R′₇, —C(CH₃)═CR₇R′₇, —CH═CR₇Hal, —C(CH₃)=CR₇Hal, and —C≡CR₇; R₅,R₆, R₇ and R′₇ each independently is H or selected from the groupconsisting of: (a) C₁-C₂₅ hydrocarbyl optionally containing one or moreheteroatoms, carbocyclic or heterocyclic moieties, and/or optionallysubstituted by one or more functional groups selected from the groupconsisting of halogen, oxo, OH, SH, CHO, NH₂, CONH₂, a negativelycharged group, and an acidic group that is converted to a negativelycharged group at the physiological pH; (b) a residue of an amino acid, apeptide or of a protein; and (c) when Y is O or S, R₅ may further be R₈⁺; m is 0 or 1; and R₈ ⁺ is H⁺ or a cation; provided that: (i) at leastone, preferably two, of R₅, R₆, R₇ and R′₇ is a hydrocarbon chain asdefined in (a) above substituted by a negatively charged group or by anacidic group that is converted to a negatively charged group at thephysiological pH; or (ii) at least one, preferably two, of R₁, R₂, andR₄ is OH, SH, O⁻R₈ ⁺ or S⁻R₈ ⁺; or (iii) at least one of R₁, R₂, and R₄is OH, SH, O⁻R₈ ⁺ or S⁻R₈ ⁺ and at least one of R₅, R₆, R₇ and R′₇ is ahydrocarbon chain substituted by a negatively charged group or by anacidic group that is converted to a negatively charged group at thephysiological pH; or (iv) at least one of R₁, R₂, and R₄ is OH, SH, O⁻R₈⁺ or S⁻R₈ ⁺ and at least one of R₅, R₆, R₇ and R′₇ is a residue of anamino acid, a peptide or of a protein; or (v) at least one of R₅, R₆, R₇and R′₇ is a hydrocarbon chain substituted by a negatively charged groupor by an acidic group that is converted to a negatively charged group atthe physiological pH and at least one of R₅, R₆, R₇ and R′₇ is a residueof an amino acid, a peptide or of a protein; but excluding the compoundsof formula I wherein M is as defined, R₃ is —C(═O)CH₃, R₁ is OH or OR₈ ⁺and R₂ is —OCH₃, and the compound of formula II wherein M is 2H, R₃ is—C(═O)CH₃, R₁, R₂ and R₄ are OH, and m is 0 or
 1. 8. Abacteriochlorophyll derivative of the formula I or II according to claim7 wherein said negatively charged groups are selected from the groupconsisting of COO⁻, cos⁻, SO₃ ⁻, and/or PO₃ ²⁻.
 9. A bacteriochlorophyllderivative of the formula I or II according to claim 7 wherein saidacidic groups that are converted to negatively charged groups at thephysiological pH are selected from the group consisting of COOH, COSH,SO₃H, and/or PO₃H₂.
 10. A bacteriochlorophyll derivative of the formulaI or II according to claim 7 wherein R₁ is Y—R₅; Y is O, S or NH; and R₅is a hydrocarbon chain substituted by functional groups selected fromOH, SH, SO₃H, NH₂, CONH₂, COOH, COSH, PO₃H₂.
 11. A bacteriochlorophyllderivative of the formula I or II according to claim 7 wherein R₅ is theresidue of an amino acid, a peptide or a protein.
 12. Abacteriochlorophyll derivative of the formula I or II according to claim1 containing a central Pd metal atom.
 13. A bacteriochlorophyllderivative of the formula I according to claim 7 wherein: M is Pd; R₁ is—NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺, —NH—(CH₂)_(n)—COO⁻R₈ ⁺; —NH—(CH₂)_(n)—PO₃ ²⁻(R₈⁺)₂; R₂ is methoxy; R₃ is —C(═O)—CH₃; R₈ ⁺ is a monovalent cation suchas K⁺, Na⁺, Li⁺, NH₄ ⁺; and n is an integer from 1 to 10, preferably 2or
 3. 14. A bacteriochlorophyll derivative of the formula II accordingto claim 7 wherein: M represents 2H, divalent Pd, Cu, or Zn or trivalentMn; R₁ is —O⁻R₈ ⁺, —NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺, —NH—(CH₂)_(n)—COOR₈ ⁺;—NH—(CH₂)_(n)—PO₃ ²⁻(R₈ ⁺)₂; or Y—R₅ wherein Y is O, S or NH and R₅ isthe residue of an amino acid, a peptide or a protein; R₂ is C₁-C₆ alkoxysuch as methoxy, ethoxy, propoxy, butoxy, more preferably methoxy; R₃ is—C(═O)—CH₃, —CH═N—(CH₂)_(n)—SO₃ ^(−R) ₈ ⁺; —CH═N—(CH₂)_(n)—COO⁻ ₈ ⁺;—CH═N—(CH₂)_(n)—PO₃ ²⁻(R₈ ⁺)₂; —CH₂—NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺;—NH—(CH₂)_(n)—COO⁻R₈ ⁺; or —NH—(CH₂)_(n)—PO₃ ²⁻(R₈ ⁺)₂; R₄ is—NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺; —NH—(CH₂)_(n)—COO⁻R₈ ⁺; —NH—(CH₂)_(n)—PO₃ ²⁻(R₈⁺)₂; R₈ ⁺ is a monovalent cation such as K⁺, Na⁺, Li⁺, NH₄ ⁺, morepreferably K⁺; and m is 1, and n is an integer from 1 to 10, preferably2 or
 3. 15. A bacteriochlorophyll derivative of formula II in claim 7wherein: M is divalent Pd; R₁ is —O⁻R₈ ⁺, —NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺, orY—R₅ wherein Y is O, S or NH and R₅ is the residue of an amino acid, apeptide or a protein; R₂ is C₁-C₆ alkoxy, preferably methoxy; R₃ is—C(═O)—CH₃, —CH═N—(CH₂)_(n)—SO₃ ⁻R₈ ⁺; or —CH₂—NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺;R₄ is —NH—(CH₂)_(n)—SO₃ ⁻ R₈ ⁺; NH—(CH₂)_(n)—COO⁻R₈ ⁺; NH—(CH₂)_(n)—PO₃²⁻(R₈ ⁺)₂; R₈ ⁺ is a monovalent cation, preferably K⁺; m is 1, and n is2 or
 3. 16. A bacteriochlorophyll derivative of the formula I accordingto claim 13, consisting of the compound Palladium bacteriopheophorbide a17³-(3-sulfopropyl)amide potassium salt.
 17. A bacteriochlorophyllderivative of the formula II according to claim 15, consisting of thecompounds: Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt;3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt; Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹,17³-di(3-sulfopropyl)amide dipotassium salt; Palladium3¹-(3-sulfopropylimino)-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹,17³-di(3-sulfopropyl)amide tripotassium salt; Copper(II)3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt; Zinc3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt; Manganese(III)3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(2-sulfoethyl)amide dipotassium salt; Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide, 17³-(N-immunoglobulin G) amide potassium salt; Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(2-carboxy-ethyl)amide dipotassium salt; Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹-(3-phosphopropyl)amide tripotassium salt; and Palladium3¹-(3-sulfopropylamino)-15-methoxycarbonylmethyl-rhodobacteriochlorin13¹,17³-di(3-sulfopropyl)amide tripotassium salt.
 18. Palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt.
 19. A pharmaceutical composition comprising abacteriochlorophyll derivative according to any one of claims 1 to 18,and a pharmaceutically acceptable carrier.
 20. The pharmaceuticalcomposition according to claim 19 for photodynamic therapy.
 21. Thepharmaceutical composition according to claim 20 for vascular-targetingphotodynamic therapy.
 22. The pharmaceutical composition according toclaim 20 or 21 for photodynamic therapy of tumors, including metastatictumors.
 23. The pharmaceutical composition according to claim 22 forphotodynamic therapy of melanoma, colon, breast, lung, or prostatecancer.
 24. The pharmaceutical composition according to claim 20 or 21for photodynamic therapy of age-related macular degeneration.
 25. Thepharmaceutical composition according to claim 20 or 21 for photodynamictherapy of benign prostate hypertrophy.
 26. The pharmaceuticalcomposition according to claim 19 for tumor diagnosis.
 27. Apharmaceutical composition according to claim 19 for killing cells orinfectious agents comprising bacteria and viruses.
 28. Thepharmaceutical composition according to claim 27 for in vitro killing ofcells or infectious agents comprising bacteria and viruses in abiological product upon illumination of said product.
 29. Thepharmaceutical composition according to claim 28 wherein said biologicalproduct is blood.
 30. Use of a compound according to any one of claims 1to 18 for the manufacture of a pharmaceutical composition for use inphotodynamic therapy.
 31. The use according to claim 30 for photodynamictherapy of tumors, including metastatic tumors.
 32. The use according toclaim 31 for photodynamic therapy of melanoma, colon, breast, lung, orprostate cancer.
 33. The use according to claim 30 for photodynamictherapy of age-related macular degeneration.
 34. Use of a compoundaccording to any one of claims 1 to 18 for the manufacture of apharmaceutical composition for diagnosis of tumors.
 35. Use of acompound according to any one of claims 1 to 18 for the manufacture of apharmaceutical composition for killing cells or infectious agentscomprising bacteria and viruses.
 36. A method for tumor photodynamictherapy which comprises: (a) administering to an individual in need acompound according to any one of claims 1 to 18; and (b) irradiating thelocal of the tumor.
 37. A method for photodynamic therapy of age-relatedmacular degeneration which comprises: (a) administering to an individualin need a compound according to any one of claims 1 to 18; and (b)irradiating the local of the macular degeneration.
 38. A method fortumor diagnosis which comprises: (a) administering to a subjectsuspected of having a tumor, a compound according to any one of claims 1to 18; and (b) irradiating the subject by standard procedures andmeasuring the fluorescence of the suspected area, wherein a higherfluorescence indicates tumor sites.
 39. In a method for photodynamictherapy using a photosensitizer, the improvement wherein saidphotosensitizer is a bacteriochlorophyll derivative according to any oneof claims 1 to
 18. 40. In a method for diagnosis of tumors using aphotosensitizer, the improvement wherein said photosensitizer is abacteriochlorophyll derivative according to any one of claims 1 to 18.41. In an in vitro method for killing of cells or infectious agentscomprising bacteria and viruses, using a photosensitizer, theimprovement wherein said photosensitizer is a bacteriochlorophyllderivative according to any one of claims 1 to
 18. 42. The compoundPalladium bacteriopheophorbide a 17³-(3-sulfo-1-oxysuccinimide) estersodium salt, as an intermediate.
 43. A method for the preparation ofcompounds of formula II In claim 7 wherein R₁ is —O⁻R₈ ⁺; R₂ is OCH₃; R₃is acetyl; R₄ is a group —NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺; R₈ ⁺ is a monovalentcation; m is 1 and n is 1 to 10, which comprises: (i) reacting thecorresponding M-bacteriopheophorbide of formula I wherein R₁ is OH withan aminosulfonic acid of the formula H₂N—(CH₂)_(n)—SO₃H in a R₈⁺-buffer; and (ii) isolating the desired compound of formula II.
 44. Themethod according to claim 43 for preparation of palladium3¹-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13¹-(2-sulfoethyl)amide dipotassium salt which comprises: (i) reactingPd-bacteriopheophorbide a with taurine of the formula H₂N—(CH₂)₂—SO₃H ina K⁺-buffer; and (ii) isolating the title compound.
 45. A method for thepreparation of compounds of formula II In claim 7 wherein R₁ is —O⁻R₈ ⁺;R₂ is —OCH₃; R₃ is acetyl; R₄ is a group —NH—(CH1 ₂)_(n)-COO⁻ R₈ ⁺; R₈ ⁺is a monovalent cation; m is 1 and n is 1 to 10, which comprises: (i)reacting the corresponding M-bacteriopheophorbide of formula I whereinR₁ is OH with an aminocarboxylic acid of the formula H₂N—(CH₂)_(n)—COOHin a R₈ ⁺-buffer; and (ii) isolating the desired compound of formula II.46. A method for the preparation of compounds of formula II in claim 7wherein R₁ is —O⁻R₈ ⁺; R₂ is —OCH₃; R₃ is acetyl; R₄ is a group—NH—(CH₂)_(n)—PO₃ ²⁻(R₈ ⁺)₂; R₈ ⁺ is a monovalent cation; m is 1 and nis 1 to 10, which comprises: (i) reacting the correspondingM-bacteriopheophorbide of formula I wherein R₁ is OH with anaminophosphonic acid of the formula H₂N—(CH₂)_(n)—PO₃H₂ in a R₈-buffer;and (ii) isolating the desired compound of formula II.
 47. A method forthe preparation of compounds of formula II in claim 7 wherein R₁ and R₄contain the same negatively charged group, which comprises: (i) reactingthe corresponding M-bacteriopheophorbide with an excess of theaminosulfonic, aminocarboxylic or aminophosphonic acid in a R₈ ⁺-buffer;and (ii) isolating the desired 13,17-disubstituted derivative of formulaII.
 48. A method for the preparation of compounds of formula II in claim7 wherein R₁ and R₄ are each a group —NH—(CH₂)_(n)—SO₃ ⁻R₈ ⁺; R₂ is—OCH₃; R₃ is acetyl; R₈ ⁺ is a monovalent cation; m is 1 and n is 1 to10, which comprises: (i) coupling the correspondingM-bacteriopheophorbide of formula I wherein R₁ is OH withN-hydroxy-sulfosuccinimide (sulfa NHS) in the presence of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC); (ii) reacting theresulting M-bacteriopheophorbide-17³-N-hydroxy-sulfosuccinimide esterwith an excess of an aminosulfonic acid of the formulaH₂N—(CH₂)_(n)—SO₃H in a R₈ ⁺-buffer, thus obtaining a compound offormula I having a sole negatively charged group at position 17; (iii)reacting the product of step (ii) with an excess of H₂N—(CH₂)_(n)—SO₃Hin a R₈ ⁺-buffer; and (iv) isolating the desired compound of formula II.49. A method for the preparation of compounds of formula II in claim 7wherein R₁ and R₄ are each a group —NH—(CH₂)_(n)—COO⁻R₈ ⁺; R₂ is —OCH₃;R₃ is acetyl; R₈ ⁺ is a monovalent cation; m is 1 and n is 1 to 10,which comprises: (i) coupling the corresponding M-bacteriopheophorbideof formula I wherein R₁ is OH with N-hydroxy-sulfosuccinimide (sulfoNHS) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC); (ii) reacting the resultingM-bacteriopheophorbide-17³-N-hydroxy-sulfosuccinimide ester with anexcess of an aminocarboxylic acid of the formula H₂N—(CH₂)_(n)—COOH in aR₈ ⁺-buffer, thus obtaining a compound of formula I having a solenegatively charged group at position 17; (iii) reacting the product ofstep (ii) with an excess of H₂N—(CH₂)_(n)—COOH in a R₈ ⁺-buffer; and(iv) isolating the desired compound of formula U.
 50. A method for thepreparation of compounds of formula II in claim 7 wherein R₁ and R₄ areeach a group —NH—(CH₂)_(n)—PO₃ ²⁻R₈ ⁺; R₂ is —OCH₃; R₃ is acetyl; R₈ ⁺is a monovalent cation; m is 1 and n is 1 to 10, which comprises: (i)coupling the corresponding M-bacteriopheophorbide of formula I whereinR₁ is OH with N-hydroxy-sulfosuccinimide (sulfo NHS) in the presence of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC); (ii) reacting theresulting M-bacteriopheophorbide-17³-N-hydroxy-sulfosuccinimide esterwith an excess of an aminophosphonic acid of the formulaH₂N—(CH₂)_(n)-PO₃H₂ in a R₈ ⁺-buffer, thus obtaining a compound offormula I having a sole negatively charged group at position 17; (iii)reacting the product of step (ii) with an excess of H₂N—(CH₂)_(n)—PO₃H₂in a R₈ ⁺-buffer; and (iv) isolating the desired compound of formula II.